Sequences displaying all of the properties expected of binding sites for -arrestins have been found in the N-terminal tails of several permeases, close to ubiquitylated lysines58,61,62. the importance of LAT1 in normal and tumor cells, little is known about the mechanisms that might control its activity, for example by promoting its downregulation via endocytosis. Here we report that in HeLa cells, activation of protein CP 376395 kinase C by phorbol 12-myristate 13-acetate (PMA) triggers efficient endocytosis and degradation of LAT1. Under these conditions we found LAT1 downregulation to correlate with increased LAT1 ubiquitylation. This modification was considerably reduced in cells depleted of the Nedd4-2 ubiquitin ligase. By systematically mutagenizing the residues of the LAT1 cytosolic tails, we identified a group of three close Rabbit Polyclonal to TOP2A (phospho-Ser1106) lysines (K19, K25, K30) in the N-terminal tail that are important for PMA-induced ubiquitylation and downregulation. Our study thus unravels a mechanism of induced endocytosis of LAT1 elicited by Nedd4-2-mediated ubiquitylation of the transporters N-terminal tail. strong class=”kwd-title” Subject terms: Endocytosis, Ubiquitylation Introduction Regulation of plasma membrane nutrient transporters is crucial for cell homeostasis. A common inhibition mechanism of these proteins involves their removal from the cell surface by selective sorting into endocytosis vesicles. Once internalized, the transporters can potentially progress along the endocytic pathway and be delivered to the lysosome, where they are degraded. This downregulation mechanism has been particularly well studied in yeast, where ubiquitin (Ub) is the signal that generally triggers transporter endocytosis1C4. This ubiquitylation is catalyzed by the Rsp5/Npi1 ubiquitin ligase, which contains a C2 domain, three WW domains, and a C-terminal catalytic domain (HECT)5C7. The WW domains typically bind to PY motifs exposed by the target proteins or -arrestin-like adaptors for Rsp5 interacting with them8,9. In mammalian cells also, Ub plays an important role in downregulating multiple plasma membrane transporters and channels10. This was initially illustrated by the epithelial Na+ channel (ENaC) in the context of the study of Liddles syndrome, a hereditary form of hypertension11. ENaC ubiquitylation involves the Nedd4-2 Ub ligase, which binds directly to PY motifs present on ENaC subunits8. Nedd4-2 is a homolog of yeast Rsp5 and one of nine members of the Nedd4 family of HECT Ub ligases9. Nedd4-type Ub ligases have since been shown to promote Ub-dependent downregulation of multiple transporters, including the dopamine transporter (DAT)12, the glutamate transporter 1 (GLT-1)13, the iron transporter (DMT1)14, the sodium-coupled neutral amino acid transporter 3 (SNAT3)15, and the cationic amino acid transporter (CAT1)16. Transporter endocytosis is often elicited by addition of PMA (phorbol 12\myristate 13\acetate), an activator of protein kinase C (PKC). The mammalian counterparts of the yeast -arrestins are the ARRestin Domain Containing (ARRDC) proteins, one of which is reported to promote endocytosis of the GLUT1 and GLUT4 glucose transporters17,18. LAT1 (L-Type amino acid transporter 1) is a bidirectional transporter of large neutral amino acids (Leu, Val, Ile, Phe, Trp, His, Met, Tyr)19C22. As one of the main transporters of several essential amino acids including leucine, LAT1 plays an important role in activating the mTORC1 (mechanistic Target of Rapamycin Complex 1) kinase complex23C28. Besides the important role of LAT1 in mTORC1 control under normal physiological conditions, for instance during T cell activation29, LAT1 is also important in CP 376395 sustaining the high metabolic demands and rapid proliferation of tumor cells22,26,30. Moreover, overexpressed LAT1 is a negative CP 376395 prognostic factor in various types of cancer, such as glioma31, renal cell carcinoma32, prostate cancer33 and breast cancer34. LAT1/SLC7A5 is a member of the SLC7 solute carrier family, which comprises two subfamilies: the cationic amino acid transporters (CATs, SLC7A1-4) and the L-type amino acid transporters (LATs, SLC7A5-11)35. LAT1 is associated, via a disulfide bridge, with the 4F2hc type II membrane glycoprotein, and this linkage is essential to the proper transport of LAT1 and its localization to the plasma membrane22,36. Recently, the tertiary structure of the human LAT1-4F2hc complex was solved by cryo-electron microscopy37. In agreement with prior predictions38C40, the 12 transmembrane segments of LAT1 were found organized in a canonical LeuT fold37. The intracellular trafficking of LAT1, and notably the mechanisms promoting its endocytosis, remain poorly known. In a recent study, we isolated a HeLa cell line stably expressing a LAT1-GFP construct. In these cells, we found LAT1 to undergo endocytosis in response to FTY72041, a sphingoid base analog that acts as an anticancer agent in animal models42. We also obtained evidence that this endocytosis results from inhibition of nutrient transport and mTORC1 inhibition, and that a similar mechanism accounts for FTY720-induced ubiquitylation and endocytosis of multiple transporters in yeast41. We now report that PKC activation by PMA induces rapid endocytosis and degradation of LAT1, that this downregulation coincides with increased ubiquitylation of LAT1, and that this modification involves the Nedd4-2.
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